Construction and identification of human SET adenovirus-delivered siRNA vector

Objective The SET/TAF-1β protein,a product of the SET gene expression,is involved in gene expression,chromatin modification,post-translational modification,and regulation of target factors at deferent levels through different pathways.This study aimed to investigate the role of the SET gene in the pathogenesis and progression of many diseases by constructing a small interfering RNA(siRNA) recombinant adenovirus vector of the human SET gene.Methods The 64nt oligonucleotide encoding human SET siRNA was inserted into the BglⅡ and HindⅢ sites of pShuttle-H1 to build a shuttle plasmid pShuttle-H1-siRNA/SET.The fragment H1-siRNA/SET of the shuttle plasmid pShuttle-H1-siRNA/SET digested by restriction enzyme NotⅠ and HindⅢ was inserted into the plasmid pAdTrack-CMV to construct a shuttle plasmid PATC-H1-siRNA/SET containing the reporter gene of the green fluorescent protein(GFP).The resultant shuttle plasmid PATC-H1-siRNA/SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant plasmid pAd-H1-siRNA/SET.The recombinant Ad-H1-siRNA/SET was packaged and amplified in the AD293 cells.The protein expression of the SET gene in the infected AD293 was detected by Western blot.Results The evidence of endonulease digestion and sequencing showed that the recombinant adenovirus vector Ad-H1-siRNA/SET was successfully constructed.The expression of GFP was observed in the AD293 cells infected with the recombinant adenovirus vector Ad-H1-siRNA/SET,and the expression the SET protein was significantly decreased(P 0.05).The inhibition rate of the SET gene expression by SiRNA was 50%-70%.Conclusion The recombinant adenovirus vector Ad-H1-siRNA/SET was successfully constructed,and its inhibitory effect on the SET expression was confirmed in the AD293 cells,which has provided a laboratory basis for further studies on the role of the SET gene in the pathogenesis and progression of many diseases.